This invention relates to a method of manufacturing a membrane device including a membrane, a membrane device, and a nanopore device having a nanopore formed in a membrane.
In order to implement a more advanced generation deoxyribonucleic acid (DNA) sequencer, a technology using a nanopore has been studied. Specifically, the technology using a nanopore involves forming a hole (nanopore) having a size comparable to that of DNA in a membrane. Further, the technology using a nanopore involves filling chambers on upper and lower sides of a thin film membrane with an aqueous solution and arranging electrodes so that the electrodes are brought into contact with the aqueous solutions in both the chambers.
The technology using a nanopore involves placing DNA to be measured in one of the chambers and applying a potential difference between the electrodes arranged in both the chambers, to thereby electrophorese the DNA. In the technology using a nanopore, a structural feature and a base sequence of the DNA are determined by measuring a change in time of an ion current flowing between the electrodes when the DNA passes through the nanopore. The technology using a nanopore is useful for acquiring structural features of various biological molecules as well as DNA.
A semiconductor substrate and a semiconductor material have high mechanical strength, and hence a nanopore device is manufactured through use of a semiconductor process. For example, in Yanagi, I., Akahori, R., Hatano, T. 86 Takeda, K. “Fabricating nanopores with diameters of sub-1 nm to nm using multilevel pulse-voltage injection” Sci. Rep. 4, 5000; DOI:10.1038/srep05000 (2014) (hereinafter referred to as “Non Patent Literature 1”), it is disclosed that a membrane is formed through use of a silicon nitride film (SiN film). Further, in Non Patent Literature 1, a fine pinhole is opened in the membrane by applying voltage stress to the membrane in an ion aqueous solution to cause puncture. The pinhole serves as a nanopore. The nanopore is also formed by etching the membrane with an aggregated electron beam.
As one of the important factors for determining DNA reading accuracy of the nanopore sequencer, there is given the thickness of the membrane. In other words, it is preferred that the thickness of the membrane be as small as possible. Each interval between adjacent bases of four kinds of bases (adenine (A), guanine (G), cytosine (C), and thymine (T)) arranged in a DNA strand is about 0.34 nanometer. As the thickness of the membrane becomes larger than the interval, a larger number of bases simultaneously enter the nanopore.
In this case, a signal obtained by current measurement is also a signal derived from a plurality of bases. Therefore, the determination accuracy of a base sequence is deteriorated, and signal analysis also becomes more complicated. Further, even when structural features of various biological molecules other than DNA are acquired, spatial resolution decreases as the thickness of the membrane becomes larger. Thus, in order to improve structure determination accuracy of an object to be measured, it is important to reduce the thickness of the membrane having a nanopore to the extent possible.
In order to reduce the thickness of the membrane, needless to say, it is preferred that a region (area) of the membrane be as narrow as possible. As the region of the membrane becomes narrower, there is a decreased probability that inevitable defects (a weak spot and a pinhole caused by, for example, a binding defect between atoms) occurring at a time of formation of the membrane are present in the membrane. Further, when the membrane is formed, it is important to avoid processes that may cause the membrane to be scraped or broken to the extent possible.
In Yanagi, I., Ishida, T., Fujisaki, K. 86 Takeda, K. “Fabrication of 3-nm-thick Si3N4 membranes for solid-state nanopores using the poly-Si sacrificial layer process” Sci. Rep. 5, 14656; doi: 10.1038/srep14656 (2015) (hereinafter referred to as “Non Patent Literature 2”), there is disclosed a method of forming a thin film SiN membrane. The forming method of Non Patent Literature 2 involves forming a thin SiN film (3 nanometers) on a Si substrate, forming a poly-Si film (150 nanometers) on the thin SiN film, and forming a SiN film (100 nanometers) on the poly-Si film. The forming method of Non Patent Literature 2 further involves opening a part of the upper SiN film, etching a rear surface of the Si substrate with a TMAH solution, and etching the poly-Si film with a KOH aqueous solution from the partially opened portion of the upper SiN film. With this, the thin film SiN membrane is formed.
The forming method of Non Patent Literature 2 does not use hydrofluoric acid at a time of forming the SiN membrane unlike Non Patent Literature 1. Therefore, in the forming method of Non Patent Literature 2, an ultrathin SiN membrane of about 3 nanometers can be formed. Further, in Non Patent Literature 2, it is disclosed that a nanopore is opened by irradiating the ultrathin SiN membrane of about 3 nanometers with an aggregated electron beam, and then a phenomenon in which DNA passes through the nanopore in an ion aqueous solution is measured based on a change in time of an ion current.
In Ashvani Kumar, Kyeong-Beom Park, Hyun-Mi Kim and Ki-Bum Kim. “Noise and its reduction in graphene based nanopore devices” Nanotechnology, 24, 495503 doi: 10.1088/0957-4484/24/49/495503 (2013) (hereinafter referred to as “Non Patent Literature 3”), as a method of reducing noise at a time of measuring an ion current flowing when the DNA passes through the nanopore, there is disclosed a method using a glass substrate. The method of Non Patent Literature 3 involves forming an amorphous Si (a-Si) film on a glass substrate, opening a part of the a-Si film by etching, opening a part of the glass substrate from a rear surface thereof, and forming a through hole so that the opened portions of the a-Si film and the glass substrate overlap each other.
The method of Non Patent Literature 3 further involves transferring by a fishing method a SiN film formed separately onto the glass substrate so as to close the opened portions and opening a pore in the SiN film. The method of Non Patent Literature 3 further involves transferring graphene onto the SiN film, forming a nanopore in the graphene, and measuring an ion current flowing when the DNA passes through the nanopore. Thus, noise at a time of ion current measurement is reduced through use of the glass substrate instead of the Si substrate.
In the forming method of Non Patent Literature 2, when the rear surface of the Si substrate is etched with the TMAH solution, one side of the thin SiN membrane (Si substrate rear surface side) is brought into contact with the TMAH solution. Further, when the poly-Si film is etched with the KOH aqueous solution from the partially opened portion of the upper SiN film, the other side of the thin SiN membrane (Si substrate upper surface side) is also brought into contact with the KOH aqueous solution. The TMAH solution and the KOH aqueous solution have characteristics of hardly etching SiN.
However, the etching amount of SiN is not completely zero. Therefore, the contact of the TMAH solution and the KOH aqueous solution with the SiN membrane damages the SiN membrane, although the damage may not be significant. In particular, when it is intended to form a thin film SiN membrane, a slight damage leads to defects of the membrane. Thus, it is required to minimize the number of times of contact of the solutions with the thin film SiN membrane at a time of wet etching.
Further, in the case of using the process of etching the poly-Si film with the KOH aqueous solution from the partially opened portion of the upper SiN film as disclosed in Non Patent Literature 2, a SiN membrane region larger than the partially opened region of the upper SiN film is formed. Actually, in Non Patent Literature 2, the partially opened region of the upper SiN film has a circular shape having a diameter of about 150 nanometers, whereas the SiN membrane region has a diameter of about 600 nanometers.
In consideration of variation in etching rate of wet etching and variation in thickness of the poly-Si film, it is not realistic that etching is stopped immediately after the SiN membrane region positioned under the poly-Si film is exposed. In other words, it is required to perform overetching in consideration of variation in etching rate of wet etching and variation in thickness of the poly-Si film. In this case, a SiN membrane region larger than the partially opened region of the upper SiN film is inevitably formed.
As described above, as the membrane region becomes narrower, there is a decreased probability that inevitable defects (a weak spot and a pinhole caused by, for example, a binding defect between atoms) occurring at a time of formation of the membrane are present in the membrane. The decreased probability is advantageous for forming the membrane. Therefore, enlargement of the SiN membrane region is disadvantageous for forming the membrane.
In Non Patent Literature 2, when an ion current flowing when the DNA passes through a nanopore is measured through use of a thin film membrane having the nanopore opened therein, noise at a time of current measurement increases. When noise at a time of current measurement is large, a current signal derived from an object to be measured becomes unclear, resulting in an increase in incorrect identification.
As one of the reasons that noise at a time of current measurement is large, there is given a large electrostatic capacitance of a structure that is sandwiched between aqueous solutions of upper and lower chambers and is formed of a membrane, a Si substrate, and a laminated film on the Si substrate. In general, as the ratio of an insulator having a low specific dielectric constant of the structure sandwiched between the aqueous solutions of the upper and lower chambers increases, the electrostatic capacitance of the structure sandwiched between the aqueous solutions of the upper and lower chambers decreases. As a result, noise at a time of measuring an ion current flowing when the DNA passes through the nanopore is reduced.
In Non Patent Literature 2, the poly-Si film is not an insulation film but a semiconductor. Therefore, only the ultrathin SiN membrane of about 3 nanometers, the upper SiN film of 100 nanometers, and the SiN film adhering to a part of the rear surface of the Si substrate are each formed of an insulation film. Under this condition, the electrostatic capacitance of the entire structure sandwiched between the aqueous solutions of the upper and lower chambers cannot be sufficiently decreased. As a result, noise at a time of measuring an ion current flowing when the DNA passes through the nanopore increases.
Further, when a SiN membrane is formed by the fishing method as in Non Patent Literature 3, the membrane is greatly damaged, and defects are liable to occur in the membrane. Further, for example, a foreign matter is liable to adhere to the membrane. Those problems cause peeling of the membrane from the a-Si film and noise at a time of measurement. Thus, stable formation of the membrane by the fishing method is difficult due to insufficient controllability. In particular, it is difficult to transfer the membrane to a wafer having a large diameter. Accordingly, it is difficult to apply the method of Non Patent Literature 3 to batch processing by a semiconductor process. In other words, the method of Non Patent Literature 3 is not suitable for formation and mass production of a membrane.